Recent studies have lead to a revised pathway for the oxidation of a cis-4 double bond to include a NADPH-dependent 2,4-dienoyl-CoA reductase step instead of the conventional epimerase pathway. Preliminary data obtained in our laboratory have indicated that the oxidation of a cis-5 double bond, encountered in the oxidation of odd-numbered double bond unsaturated fatty acids, is not through the conversion to a cis-3 enoyl-CoA, followed by isomerization to a trans-2-enoyl-CoA, as it appears in all biochemistry textbooks. Instead, it is through a direct reduction of the cis-5-enoyl-CoA to its saturated analogue via a NADPH-dependent pathway. Therefore, the oxidation of all unsaturated fatty acids requires a reduction step which in dependent on the availability of NADPH. In this proposal, we will study the existence of this reduction pathway in intact mitochondria and peroxisomes from rat liver and other organs. The reductase will be isolated and purified. This enzyme will be compared to 2,4-dienoyl-CoA reductase. In addition, he mechanism of reduction and the metabolic block of a cis-5 double bond to beta-oxidation will be studied with synthetic intermediates and with stable isotope labels. The effect of double bond on the oxidation of fatty acids will be studied with oxygen electrode and gas chromatograph-mass spectrometry. Also, these effects will be studied at enzyme level with acyl-CoA dehydrogenases, crotonases, and 3-hydroxyacyl-CoA dehydrogenases. Potential "mechanism-based" inhibitors to this reduction pathway will be investigated with analogues containing acetylene group. This newly revised pathway for the metabolism of unsaturated fatty acids explains the high activity of transhydrogenase, which converts NADP to NADPH, in heart cells where no significant synthetic function is known. In Addition, this reduction pathway might be an universal pathway for the metabolism of other cis-5 containing endogenous substrates, which include prostaglandins and related metabolites. The knowledge derived from this investigation will guide us in the evaluation of patients with inborn errors of fatty acid metabolism for a possible reductase deficiency. Whether the control of the reductase activity or the level of NADPH is related to cancer promotion or the beneficial effects on lipoproteins, attributed to unsaturated fatty acids, remains to be investigated.